Evaluation of Morphological, Qualitative, and Metabolomic Traits during Fruit Ripening in Pomegranate (Punica granatum L.)

Round 1

Reviewer 1 Report

The manuscript carried out morphological and chemical characterization of three Italian germplasm accessions ("Santa Lucia", "Di Benedetto" and "arborea") to evaluate the differences in the changes of physicochemical and nutritional traits in the four stages of fruit ripening. However, the manuscript still has the following problems:

Major Problems:

1.The manuscript studied three pomegranate materials, please explain why these three materials are selected and their characteristics.

1.The manuscript does not introduce the antioxidant activity of pomegranate. The antioxidant activity of pomegranate is indeed mentioned in the results. Please add why the antioxidant activity of pomegranate is measured and what impact the antioxidant activity has on the development of pomegranate.

3.In result “Bioactive Compounds and Antioxidant Activity”, there are many descriptions of other literature results, which makes this part of the results look a little confused. It is suggested to modify this part of the content to make the content clearer.

4.In the abstract part of the manuscript, there is no description of research significance. It is suggested to add relevant content.

5.In the introduction of the manuscript, there are few descriptions of the four stages of pomegranate fruit ripening and the effects of bioactive compounds. It is suggested to supplement relevant contents.

Minor Problems:

6.For the table format in the manuscript, it is recommended to use the three line table format.

7.The picture in Figure 3 is of poor quality. It is suggested to replace the clear picture to make the reader see more clearly.

8.The format of references is not uniform, such as references 18 and 23. It is recommended to check the format of all references to make them uniform.

Author Response

REVIEWER 1

The manuscript carried out morphological and chemical characterization of three Italian germplasm accessions ("Santa Lucia", "Di Benedetto" and "arborea") to evaluate the differences in the changes of physicochemical and nutritional traits in the four stages of fruit ripening. However, the manuscript still has the following problems:

Major Problems:

Point 1.The manuscript studied three pomegranate materials, please explain why these three materials are selected and their characteristics.

Answer 1: These autochthons pomegranate varieties have been selected by farmers, but they have never been studied for agronomic and nutraceutical traits, therefore the differences between the physico-chemical characters of the fruits were studied.

Point 2. The manuscript does not introduce the antioxidant activity of pomegranate. The antioxidant activity of pomegranate is indeed mentioned in the results. Please add why the antioxidant activity of pomegranate is measured and what impact the antioxidant activity has on the development of pomegranate.

Answer 2. Dear reviewer, thank you for your suggestion. In lines 44-50 have been added these informations: “Some bioactive compounds are present in pomegranate fruits, that is compounds with or without nutritional value and endowed with biological activity that is expressed in reducing the risk of developing numerous chronic diseases, thus carrying out a fundamental protective action on the health of consumers. The importance of the antioxidants contained in fruits is to be associated with the beneficial effects that they play in the human body against chronic-degenerative diseases induced by oxidative stress and age”.

 Point 3. In result “Bioactive Compounds and Antioxidant Activity”, there are many descriptions of other literature results, which makes this part of the results look a little confused. It is suggested to modify this part of the content to make the content clearer.

Answer 3. This section has been changed.

Point 4. In the abstract part of the manuscript, there is no description of research significance. It is suggested to add relevant content.

Answer 4. The purpose of the research was explained in the lines 20- 23: “The aim of this study was to evaluate the changes in physico-chemical and nutritional traits of three Italian germplasm accessions (‘Santa Lucia’, ‘Di Benedetto’ and ‘Arborea’) to determine the differences of these parameters both within the same cultivar during the four ripening stages and between the individual cultivars in the same ripening stage”.

Point 5. In the introduction of the manuscript, there are few descriptions of the four stages of pomegranate fruit ripening and the effects of bioactive compounds. It is suggested to supplement relevant contents.

Answer 5. Dear reviewer thank you for your consideration. At lines 75-80 this information has been added: “The colour changes of skin are due to anthocyanins synthesis during ripening process. These water soluble vacuolar pigments are members of the phenolic compounds that contribute to the red, blue or purple colors of many fruits, including pomegranate skin and seed, and they are well known for their antioxidant activity. The total anthocyanin content increase significantly during fruit ripening”

At lines 88-95 this information has been added: “The fruits were collected at four ripening stages, taking into account the color of the fruit skin: immature, un-ripe, semi-ripe and ripe fruit. The immature stage is characterized by pome-granates with green skin color and white seeds, the un-ripe stage in which the skin color is a light red epicarp with light-pink colour seeds, semi-ripe stage pomegranates with red epicarp and light-red seeds, and mature stage includes ripe pomegranates with intense red skin colour and mature red seeds. The four samplings were made from September 3 to November 3 (Table 1).”.

Minor Problems:

Point 6. For the table format in the manuscript, it is recommended to use the three line table format.

Answer 6. The table format has been changed

Point 7. The picture in Figure 3 is of poor quality. It is suggested to replace the clear picture to make the reader see more clearly. FIORENTINO

Answer 7. Picture quality has been improved

Point 8. The format of references is not uniform, such as references 18 and 23. It is recommended to check the format of all references to make them uniform

Answer 8. All references have been uniformed

Author Response File: Author Response.pdf

Reviewer 2 Report

The aim of the manuscript ‘horticulturae-1678490’ is to carry out a morpho-pomological and chemical characterization of  three Italian germplasm accessions (‘Santa Lucia’, ‘Di Benedetto’ and ‘Arborea’), to evaluate their differences in changes in physico-chemical and nutritional traits during four  stages of fruit ripening.

Generally, it is well written and informative, concise paper. Used basic methods allow to get proper results. However, in my opinion there is a lack of information about specific ripening stages. I wonder on what basis was the stage of the ripening process determined? Were measurements of the level of e.g. ethylene taken? How did the authors assess the stage of the ripening process? Only the color of fruit decided? I understand that the parameters of the fruit have been determined but the exact stages of ripening are not specified enough.

Overall, due to the very significant importance of agricultural research, the manuscript is acceptable for publication. However, this studies should be completed with mentioned information. Then, in my opinion the manuscript can be accepted for publication as a first short report about ripening proces in Pomegranate fruit.

Author Response

REVIEWER 2

The aim of the manuscript ‘horticulturae-1678490’ is to carry out a morpho-pomological and chemical characterization of  three Italian germplasm accessions (‘Santa Lucia’, ‘Di Benedetto’ and ‘Arborea’), to evaluate their differences in changes in physico-chemical and nutritional traits during four  stages of fruit ripening.

Generally, it is well written and informative, concise paper. Used basic methods allow to get proper results. However, in my opinion there is a lack of information about specific ripening stages. I wonder on what basis was the stage of the ripening process determined? Were measurements of the level of e.g. ethylene taken? How did the authors assess the stage of the ripening process? Only the color of fruit decided? I understand that the parameters of the fruit have been determined but the exact stages of ripening are not specified enough. Overall, due to the very significant importance of agricultural research, the manuscript is acceptable for publication. However, this studies should be completed with mentioned information. Then, in my opinion the manuscript can be accepted for publication as a first short report about ripening proces in Pomegranate fruit.

Dear reviewer thank you for your consideration, below is the explanation: “The fruits were collected at four ripening stages, taking into account the color of the fruit skin: immature, un-ripe, semi-ripe and ripe fruit. The immature stage is characterized by pome-granates with green skin color and white seeds, the un-ripe stage in which the skin color is a light red epicarp with light-pink colour seeds, semi-ripe stage pomegranates with red epicarp and light-red seeds, and mature stage includes ripe pomegranates with intense red skin colour and mature red seeds. The four samplings were made from September 3 to November 3 (Table 1).”. This information was added from line 88-95.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The manuscript carried out morphological and chemical characterization of three Italian germplasm accessions ("Santa Lucia", "Di Benedetto" and "arborea") to evaluate the differences in the changes of physicochemical and nutritional traits in the four stages of fruit ripening. In the revised manuscript, the author explained why the three pomegranate materials were selected for research, supplemented other content problems, and modified the pictures and format. The revised manuscript is relatively complete, so I recommend accepting it in its current form.

Author Response

the English language and style have been improved

Author Response File: Author Response.docx

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Pomegranate is an attractive fruit with excellent taste and high bioactive value, and its consumption and market demanding are rising. There are several accessions in Italy and cultivars widespread in different countries with various bio-agronomic features. Among the others, fruit features are of the most valuable ones. And several groups of authors have characterized the fruit features during ripening for some accessions outside Italy. In the present study, “Evaluation of Morphological, Qualitative and Metabolomic Traits During Fruit Ripening in Pomegranate (Punica granatum L.)” Cirillo et al. evaluated the feathers of 3 Italian pomegranate accessions during fruit ripening. The authors not only present the feathers of the 3 accessions, but also compared with other accessions by other authors. Besides the classical parameters such as fruit weight, size, color and nutrition values, etc. the authors applied the metabolomic analysis, which may be a trend in the future for the high throughput evaluation of the similarity or dissimilarity of the metabolsome between the crop accessions. Overall, the results are clearly presented and the manuscript can be accepted for published only after some concerns are clarified and some mistakes are fixed.

1. The authors present reasonable statistics analysis in the figures and tables. But, in the experiment design, the author only described that 30 fruits were randomly collected from 5 trees. How many biological repeats for each data? How the authors clarified as one biological repeat?
2. In table 3, how to explain there are significant changes in POL and FLA, but no significant changes in ANT? It may be more interesting to present the correlation and interaction between these bioactive values as the other features (Table S1-3).
3. It is not clear what the significance means in table 2 and 3.
4. In Fig. 3, there is no description and data for JC.
5. In all the tables, including the supplementary tables, different letters indicate significant difference in the same column but not the same row.
6. In line 425-426, there is no description for the data “MS” and “A”
7. some sentences highlighted in the PDF is not clear or with grammar mistakes.

Comments for author File: Comments.pdf

Author Response

Pomegranate is an attractive fruit with excellent taste and high bioactive value, and its consumption and market demanding are rising. There are several accessions in Italy and cultivars widespread in different countries with various bio-agronomic features. Among the others, fruit features are of the most valuable ones. And several groups of authors have characterized the fruit features during ripening for some accessions outside Italy. In the present study, “Evaluation of Morphological, Qualitative and Metabolomic Traits During Fruit Ripening in Pomegranate (Punica granatum L.)” Cirillo et al. evaluated the feathers of 3 Italian pomegranate accessions during fruit ripening. The authors not only present the feathers of the 3 accessions, but also compared with other accessions by other authors. Besides the classical parameters such as fruit weight, size, color and nutrition values, etc. the authors applied the metabolomic analysis, which may be a trend in the future for the high throughput evaluation of the similarity or dissimilarity of the metabolome between the crop accessions. Overall, the results are clearly presented, and the manuscript can be accepted for published only after some concerns are clarified and some mistakes are fixed.

• The authors present reasonable statistics analysis in the figures and tables. But, in the experiment design, the author only described those 30 fruits were randomly collected from 5 trees. How many biological repeats for each data? How the authors clarified as one biological repeat?                    Dear reviewer, thank you for your considerations. “The morphological parameters of the fruits were carried out on all 30 fruits collected, while, the chemical analyzes carried out on the pomegranate juice were carried out on 5 repeats and each biological repeat consisted of 3 fruits”. This explanation has been added in the text to the lines 88-91.
• In table 3, how to explain there are significant changes in POL and FLA, but no significant changes in ANT? It may be more interesting to present the correlation and interaction between these bioactive values as the other features (Table S1-3).                                                                                      Dear reviewer, thank you for your report. The data reported in Table 3 has been shown in Fig.3, highlighting the differences among different bioactive compounds during ripening stages in pomegranate cultivars. Correlations have been carried out in preliminary PCA analysis without significant interactions (data not shown).
• It is not clear what the significance means in table 2 and 3.                "Significance means " indicates the significant statistical difference of the various parameters measured   during ripening for all three cultivars studied.  ns= not significant ;  * = 0.01 ≤ p ≤0.05;  ** = 0.001≤ p ≤ 0.01; *** = p ≤ 0.001. This explanation has been added in the text, below the tables.
• In Fig. 3, there is no description and data for JC.                                      Dear reviewer, thank you for your report. The authors added the abbreviation JC = juice content (%) and the data were reported in table 2.
• In all the tables, including the supplementary tables, different letters indicate significant difference in the same column but not the same row.     Thanks for reporting, the authors corrected the error, the significant difference is only in the same column.
• In line 425-426, there is no description for the data “MS” and “A”.          “MS” and “A” have been changed in the revised manuscript.
• Some sentences highlighted in the PDF is not clear or with grammar mistakes.                                                                                                      The authors modified the sentences as requested by the reviewer.

• Line 39: Lythraceae, subclass Rosidae not italic;
• In line 218-225 the sentences have been changed to“Arils weight recorded an increasing trend during the three ripening stages in all accessions, in particular, the highest values were recorded in 'Santa Lucia' with an average of 137.81g showing at IV stage an increase of 74.04 %. ‘Di Benedetto’ and ‘Arborea’ accessions showed an average of 39g and 110.73g ,  showing an  increase of arils weight of 76.42 % and 83.89 % respectively, from I to IV stages. 'Di Benedetto' and 'Arborea' reported a juice content of 70% and 69.50% at stage IV, respectively, while 'Santa Lucia' showed a juice content of 53.25% (Table 2)”
• In line 236 the sentences have been changed to: “it is important to highlight optimizing the quality attributes of these fruit parts is the goal of growers, breeders and processors”.
• Line 367 The sentence has been corrected and changed to “The accumulation of sugars during ripening stages is directly controlled by increasing the activities of sucrose synthase and sucrose phosphate synthase”
• Line 370 The sentence has been changed to “The accumulation of organic acids, such as citric and malic acids, in mesocarp cells depends both on the genotype and on stresses caused by the environment”
• Line 371 The sentence has been corrected to “Different organic acids, the most abundant being citric and malic acids, are reported from fruits, leaves and seeds of pomegranate.”
• Line 424
• “MS” and “A” have been changed in the revised manuscript.

Author Response File: Author Response.docx

Reviewer 2 Report

First of all, I would like to congratulate the authors for their work. 

The article focuses on an important topic, the characterization of regional fruit tree cultivars. These cultivars are an important genetic resource and can be valued for their specificities and can contribute to the development of the regions where they are cultivated. However, this article raises a question that would need to be answered in order to analyze the data. Do these 3 cultivars have the same maturation time? On each of the sampling dates, were the fruits in the same state of maturity?

On the other hand, does it make sense to compare cultivars by mixing data from each of them at different states of maturation? What is really comparable are the characteristics of each of the cultivars in the optimal state of maturity of each one of them.

What is the meaning of the average soluble solids content (for example) of immature fruits (unfit for consumption) with ripe fruits, harvested two months later? How important or useful is this average value? For example (in table S1), on average, 'Santa Lucia' has a juice content of 54.35%, higher than that of the 'Arborea' cultivar, with only 37.48%. However, in the maturation state (sampling IV) 'Arborea' has a higher percentage (69.50) of juice than 'Santa Lucia' (53.25). Which one is the juiciest one? The authors consider 'Arborea the least juicy (line 213). I would say otherwise. What matters is the juice content of the ripe fruit. Therefore, 'Santa Lucia' is the least juicy and ‘Arborea’ is the best one in this parameter.

I now add some specific comments:

Line 67: The fruits can be destined for the fresh market (with or without cold storage) or for industrial processing.

Line 78: “ to evaluate their differences in changes in physico-chemical and nutritional traits”. It seems that the authors wanted to assess the differences between cultivars and the evolution of each one of them.

Line 87: … randomly harvested from five pomegranate …

Line 111: “peel” and “pith” are not the most appropriate terms.

Line 120: The acidity of fruit juice is not usually expressed as "g citric acid L⁻¹", but rather as "g citric acid 100 mL⁻¹". This has consequences for the maturity index values (TSS/TA). All data presentation and discussion should be reviewed.

Line 127: This color index was not proposed by Pathare et al, but by Jiménez-Cuesta et al. (1981). Pathare et al. only reviewed the topic.

Line 156: “NMR”- As in other cases, the first time the abbreviation appears, it must be accompanied by its meaning.

Line 191: The first part of table S1 must be included in the article. It could even be included almost the entire table, excluding data presented in figure 1.

Line 230: The acidity of the cultivar 'Santa Lucia' is really very low. Values will have to be confirmed and, if they are real, it is necessary to further discuss this characteristic of the cultivar.

Line 232: Figure 1B; 1D)

Figure 1: Graphs B and C must be redone with the new units

Table 2; line 282: The value “116.87 ± 76.83 a” seems very strange. Both the mean and the standard error are very different from the other values. Wasn't there some failure in some measurement?

Table 2 and 3: The values presented in tables 2 and 3 would be easier to understand if they were as graphs, following the example in figure 1.

Line 402: "PCA analysis was …" contains a repetition of "analysis", abbreviated, and spelled out. It would be better “Principal component analysis (PCA) was ...”

General comment on "PCA analysis": This statistical tool is important, but I do not identify its usefulness in this case. Proving that the 3 cultivars are different is no big deal. A graphical representation of the amount of bioactive compounds of each cultivar in its optimal state of maturation would be much more interesting.

I would encourage the authors to improve the article. A better presentation of results will make it possible to value the work performed.

Author Response

First of all, I would like to congratulate the authors for their work. 

The article focuses on an important topic, the characterization of regional fruit tree cultivars. These cultivars are an important genetic resource and can be valued for their specificities and can contribute to the development of the regions where they are cultivated. However, this article raises a question that would need to be answered in order to analyze the data.

• Do these 3 cultivars have the same maturation time? On each of the sampling dates, were the fruits in the same state of maturity?

        Dear reviewer, the authors did not know these three pomegranate accessions analyzed and have chosen to analyze the color of the epicarp of the fruits in the 4 different coloring stages shown in table 1. From the data obtained it is shown that the three accessions show a trend of the coloring of the epicarp different (for example the Di Benedetto accession presents values of a * greater). The authors, therefore, chose these accessions to carry out a characterization of the local biodiversity of pomegranate.

• On the other hand, does it make sense to compare cultivars by mixing data from each of them at different states of maturation? What is really comparable are the characteristics of each of the cultivars in the optimal state of maturity of each one of them. What is the meaning of the average soluble solids content (for example) of immature fruits (unfit for consumption) with ripe fruits, harvested two months later? How important or useful is this average value?

         The authors wanted to evaluate the variation of the chemical-physical characteristics over time in pomegranate fruits to evaluate the optimal harvesting period in which the fruits had better qualitative characteristics (which in our case was the III stage).

• For example (in table S1), on average, 'Santa Lucia' has a juice content of 54.35%, higher than that of the 'Arborea' cultivar, with only 37.48%. However, in the maturation state (sampling IV) 'Arborea' has a higher percentage (69.50) of juice than 'Santa Lucia' (53.25). Which one is the juiciest one? The authors consider 'Arborea the least juicy (line 213). I would say otherwise. What matters is the juice content of the ripe fruit. Therefore, 'Santa Lucia' is the least juicy and ‘Arborea’ is the best one in this parameter.

         Dear reviewer, thank you for your comment, the authors changed this sentence to: 'Di Benedetto' and 'Arborea' reported a juice content of 70% and 69.50% at stage IV, re-spectively, while 'Santa Lucia' showed a juice content lower of 53.25% (Table 2), line 220-222.

• I now add some specific comments:
• Line 67: The fruits can be destined for the fresh market (with or without cold storage) or for industrial processing.

       Dear reviewer thanks for this consideration, it has been added at line 66-68.

• Line 78: “to evaluate their differences in changes in physico-chemical and nutritional traits”. It seems that the authors wanted to assess the differences between cultivars and the evolution of each one of them.

          Dear reviewer, as reported in the text at the line 79-82, the aim of our study was to evaluate the evolution of chemical-physical and nutritional changes in the four ripening stages for all three accessions considered.

• Line 87: … randomly harvested from five pomegranate …

The word has been changed in line 88.

• Line 111: “peel” and “pith” are not the most appropriate terms.

The words have been changed with “epicarp and carpellary membranes” in line 118.

• Line 120: The acidity of fruit juice is not usually expressed as "g citric acid L⁻¹", but rather as "g citric acid 100 mL⁻¹". This has consequences for the maturity index values (TSS/TA). All data presentation and discussion should be reviewed.

       Thanks for your comment, the authors modified the acidity results in citric acid values 100 mL ^{− 1} and recalculated the TSS / TA ratio (Figure 2).

• Line 127: This color index was not proposed by Pathare et al, but by Jiménez-Cuesta et al. (1981). Pathare et al. only reviewed the topic.

 Dear reviewer, the authors recalculated the pomegranate color index using a new formula:

L *·a *·b * ⁻¹ proposed by Manera et al. 2013. (line 136).

• Line 156: “NMR”- As in other cases, the first time the abbreviation appears, it must be accompanied by its meaning.

      The abbreviation is now defined the first time it appears in the text

•  Line 191: The first part of table S1 must be included in the article. It could even be included almost the entire table, excluding data presented in figure 1.

         Dear reviewer, thank you for your advice, the authors have inserted table S1 in the article at line 248.

• Line 230: The acidity of the cultivar 'Santa Lucia' is really very low. Values will have to be confirmed and, if they are real, it is necessary to further discuss this characteristic of the cultivar.

       The authors confirm that the acidity of Santa Lucia accession is very low, and this characteristic will be investigated in further studies.

• Line 232: Figure 1B; 1D)

    The following changes were made to the line 283.

• Figure 1: Graphs B and C must be redone with the new units

       The graphics have been changed.

• Table 2; line 282: The value “116.87 ± 76.83 a” seems very strange. Both the mean and the standard error are very different from the other values. Wasn't there some failure in some measurement?

         Thanks for your correction, the authors checked and edited the error in figure 2B.

• Table 2 and 3: The values presented in tables 2 and 3 would be easier to understand if they were as graphs, following the example in figure 1.

     Dear reviewer, thank you for your advice. The authors replaced Tables 2 and 3 with figures,    following the example in Figure 1 (Figure 2 and 3).

• Line 402: "PCA analysis was …" contains a repetition of "analysis", abbreviated, and spelled out. It would be better “Principal component analysis (PCA) was ...”

           The sentence has been changed to the line 456.

General comment on "PCA analysis": This statistical tool is important, but I do not identify its usefulness in this case. Proving that the 3 cultivars are different is no big deal. A graphical representation of the amount of bioactive compounds of each cultivar in its optimal state of maturation would be much more interesting.PCA analysis confirms the diversity among three pomegranate cultivars. In the same figure has been reported score plot and loading plot to discriminate the different cultivars on the basis of analyzed variables. Furthermore, a graphical representation of the amount of bioactive compounds of each cultivar in its optimal state of maturation (Fig.3)

I would encourage the authors to improve the article. A better presentation of results will make it possible to value the work performed.

Author Response File: Author Response.docx

Reviewer 3 Report

The article presents a confusing and partial structure, with most of the relevant data/results presented as "Supplementary material".

The authors use wrong terms to refer to the pomegranate parts since they used  "aril" when referring to the "pomegranate seed". Note that the pomegranate is an exarilated fruit. Please check and consult https://doi.org/10.1016/j.tifs.2020.02.014.

Why does the author use “pith” to refer to the “carpellary membranes”?. Please correct it.

Please, check the numerical citations of the results in the text, some lack the unit and for others, the authors need to change the "comma" for a "point" (e.i. Ln 322).

The authors mix results with discussion and vice versa. Please, following the journal format, separate “Results” and “Discussion”

The methodology presents important gaps, making its replicability impossible, and generating many doubts, some such as:

1. Why did the authors choose these pomegranate varieties?
2. What are the irrigation conditions and management of the crops?
3. Why did the authors choose those collection dates/ripening stages? Please justify the ripening stages studied and specify the time elapsed since blossom.
4. Please, specify the temperature in all the experimental phases since some methodologies used have a "temperature correction index" (e.i. TSS).
5. The juice sample was obtained from 100g of seed? Was enough juice for analysis?
6. Why do the authors use the colourimetric index in pomegranate? Since it is calculated based on a specific formula for citrus fruits! Some modifications were made for other fruits like pomegranate: https://doi.org/10.1016/j.scienta.2012.11.036
7. Bioactive Compounds and Antioxidant Activity - Although the bibliographic reference is given, please, briefly explain the methodology to help the reader understand the work in an integral way.

Where are the 1H NMR Analysis results? Where is the sample's spectrum? and the metabolites studied? The 1H NMR PCA is a complementary non-substitute results/spectrum tool.

Both the results and the discussion are poor, partial and confusing. The authors do not analyze the variations between the different states for each variety. As expected, modifications in the physical and chemical characteristics of the pomegranates are necessary, but the authors do not evaluate the differences and intrinsic particularities for each pomegranate variety.

The conclusions presented are very general and are not aligned with the results/discussion presented in the work.

Most of the references are old. I should recommend updating it since a lot of research on pomegranate has been published in recent years.

Author Response

• The article presents a confusing and partial structure, with most of the relevant data/results presented as "Supplementary material".                Dear reviewer, following your comment the authors, have included other data in the manuscript (Table 2) and they deleted some supplementary tables.

• The authors use wrong terms to refer to the pomegranate parts since they used "aril" when referring to the "pomegranate seed". Note that the pomegranate is an exarilated fruit. Please check and consult https://doi.org/10.1016/j.tifs.2020.02.014.

Dear reviewer we have read this interesting paper and the Authors have changed the term "aril" into "pomegranate seed" in whole paper.

• Why does the author use “pith” to refer to the “carpellary membranes”?. Please correct it.

The word was changed to line 118.

• Please, check the numerical citations of the results in the text, some lack the unit and for others, the authors need to change the "comma" for a "point" (e.i. Ln 322).

Done

• The authors mix results with discussion and vice versa. Please, following the journal format, separate “Results” and “Discussion”.

Dear reviewer, the authors have checked, and the journal accepts the format with mix of results and discussions.

• The methodology presents important gaps, making its replicability impossible, and generating many doubts, some such as:
• Why did the authors choose these pomegranate varieties?

Dear reviewer, the authors did not know these three pomegranate accessions analyzed and have chosen to analyze the color of the epicarp of the fruits in the 4 different coloring stages shown in table 1. From the data obtained it is shown that the three accessions show a trend of the coloring of the epicarp different (for example the Di Benedetto accession presents values of a * greater). The authors, therefore, chose these accessions to carry out a characterization of the local biodiversity of pomegranate.

• What are the irrigation conditions and management of the crops?

The plants were trained to free vase systems and spaced 4.0 m between the rows and 3.5 m on the row with a planting density of 714 trees ha^-1. The experiment was carried out on medium-textured soil with an adequate content of macro and micro elements. Irrigation was provided using a drip system equipped with two self-compensating drippers for plant, delivering 8 L/h. The orchard received standard horticultural cares and the treatments against the main parasites have been established in accordance with the regulation governing integrated production. This sentence has been adding in line 89-95

• Why did the authors choose those collection dates/ripening stages? Please justify the ripening stages studied and specify the time elapsed since blossom.

The authors choose those sampling dates based on the epicarp color variation of the 3 accessions studied. Sorry but we haven't any date on bloom of pomegranate.

• Please, specify the temperature in all the experimental phases since some methodologies used have a "temperature correction index" (e.i. TSS).

The TSS determination was made using a refractometer (Atago, model PR-101a, Tokyo, Japan) with a Temperature compensation system.

• The juice sample was obtained from 100g of seed? Was enough juice for analysis?

Sorry, the juice sample was obtained from 300g of seed (line 119)

• Why do the authors use the colourimetric index in pomegranate? Since it is calculated based on a specific formula for citrus fruits! Some modifications were made for other fruits like pomegranate: https://doi.org/10.1016/j.scienta.2012.11.036

Dear reviewer, the authors recalculated the pomegranate color index using the formula L * · a * · b * −1 proposed by Manera et al. 2013

• Bioactive Compounds and Antioxidant Activity - Although the bibliographic reference is given, please, briefly explain the methodology to help the reader understand the work in an integral way.

These information has been added in the revised manuscript.

• Where are the 1H NMR Analysis results? Where is the sample's spectrum? and the metabolites studied? The 1H NMR PCA is a complementary non-substitute results/spectrum tool.

The usual protocol for NMR based metabolomics consists in the multivariate data analysis of the data in order to evidence the metabolites that are responsible for the separation, in order to focus then on their identification, with no need to identify the compounds that are instead the same and in comparable relative amounts in all of the samples. Since the aim of this work is to evidence the differences among the cultivars during ripening time, this approach was here applied. Therefore, PCA is the best way to visualize these data. It would be really impractical and difficult to evidence these results by analysing the single NMR spectra. In order to make it easier to follow the discussion, a proton NMR spectrum has now been submitted as supplementary figure and the signals generated by the metabolites responsible for the separation (as discussed in the manuscript) are evidenced on the spectrum.

• Both the results and the discussion are poor, partial and confusing. The authors do not analyze the variations between the different states for each variety. As expected, modifications in the physical and chemical characteristics of the pomegranates are necessary, but the authors do not evaluate the differences and intrinsic particularities for each pomegranate variety.

Results and discussion have been improved in the revised manuscript.

• The conclusions presented are very general and are not aligned with the results/discussion presented in the work.

Conclusions have been modified in the revised manuscript.

• Most of the references are old. I should recommend updating it since a lot of research on pomegranate has been published in recent years.

Other recent references have been added in the revised manuscript and the numbers of reference list have been changed.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The article continues to have several errors. The abstract does not agree with the results presented. This must imperatively be corrected, so as not to discredit the journal and the authors. I strongly advise authors to take this (last) opportunity to review the text to verify the values presented. Some leave doubts. Please take the opportunity to check the entire text, so that there are no errors like those pointed out in the attached file.

Comments for author File: Comments.pdf

Author Response

Fruit pigmentation can increase, but Morphological traits don't increase; they can change.

Dear reviewer, as you suggested the authors have changed the word “increase” in “a variation” line 22

Do you really think it's interesting to give the value to the hundredth of a gram? Especially when the standard error is 19 g...

As you suggested, the authors changed the value to: 392.2 at line 24 and, the last decimals have been eliminated throughout the text.

Arborea showed the almost the same juice content: 70,0 and 69,5 are not statistically different, so you can´t say that Di Benedetto showed the highest value.

The sentence has been changed at line 23: " The highest fruit weight was detected in ‘Di Benedetto’ (392.19 g) that also with 'Arborea' displayed high juice content”

At Line 52 the authors have changed “ripen” to “harvested”

What I wrote was a comment that should have led the authors to realize that what was written was wrong and that they should rewrite the sentence. The authors simply copied my comment into the sentence, leaving a meaningless sentence.

Dear reviewer, the authors have rephrased the sentence: “As for other species [14,15], even for pomegranate fruits, by identifying the optimal moment of harvesting, it is possible to allocate them for direct consumption or for transformation” (line 65-67).

It does not seem reasonable that the fruits have decreased in size from phase III to phase IV. Probably, in one of these phases, the sampling focused on fruits that were not representative of that phase and those trees. This may have affected the results in other parameters. For example, on the TSS descent.

Dear reviewer, during different ripening phases, the fruits were sampled evaluating the sample on each plant trying to realize a representative sampling of each phase and each trees. The sampling for each phase is destructive and it is not possible to follow the growth trend of the same fruit on trees.

The last decimal place could be eliminated in all columns except one

It has been done

TA (g citric ....)

It has been added

Author Response File: Author Response.docx

Reviewer 3 Report

The authors have done and answered all my suggestions/comments, and the manuscript was modified satisfactorily.

Author Response

Dear reviewer, thank you for your suggestions